This invention relates to oligonucleotides modified by a pendant cholesteryl group. More particularly, the present invention is related to a cholesteryl modified oligonucleotide and a method of using the modified oligonucleotide as an antiviral agent.
The pioneering work of Zamecnik and Stephenson, Proc. Natl. Acad., 75:280-284 (1978), on antiviral activity of oligonucleotides and Miller and Ts'o, on the chemistry and biochemistry of non-ionic analogues (Barrett, et al., Biochem., 13:4898-5 (1974) and Jayaraman, et al. Proc. Natl. Acad Sci. U.S.A., 78:1537-1541 (1981)) has stimulated extensive research directed the therapeutic potential of nucleotide polymers. Oligonucleotide analogues with methylphosphonate, Miller, et al., Biochemie, 67: 769-776 (1985), Agris, et al., Biochem., 25:6268-6275 (1986), Smith, et al., Proc. Natl. Acad. Sci. U.S.A., 83:2787-2791 (1986), and Sarin, et al., Proc. Natl. Acad. Sci. U.S.A., 85:7448-7451 (1988); phosphorothioate, Matsukura, et al., Proc. Natl. Acad. Sci. U.S.A., 84:7706-7710 (1987) and Agrawal, et al., Proc. Natl. Acad. Sci. U.S.A., 85:7079-7083 (1988); and phosphoramidate, Agrawal, et al., Proc. Natl. Acad. Sci. U.S.A., 85:7079-7083 (1988), backbones as well as natural type oligonucleotides Zamecnik, et al., Proc. Natl. Acad. Sci. U.S.A., 83:4143-4146 (1986), and a polylysine conjugate, Goodchild, et al., Proc. Natl. Acad. Sci. U.S.A., 85:5507-5511 (1988), have now been reported to inhibit viral replication in cell culture. The viruses studied in this context include Rous sarcoma virus, Zamecnik and Stephenson, Proc. Natl. Acad., 75:280-284 (1978); simian virus, Miller, et al., Biochemie, 67: 769-776 (1985); vesicular stomatitis virus, Agris, et al., Biochem., 25:6268-6275 (1986) and Lemaitre, et al., Proc. Natl. Acad. Sci. U.S.A., 84:648-652 (1987); human immunodeficiency virus (HIV), Sarin, et al., Proc. Natl. Acad. Sci. U.S.A., 85:7448-7451 (1988), Matsukura, et al., Proc. Natl. Acad. Sci. U.S.A., 84:7706-7710 (1987), Agrawal, et al., Proc. Natl. Acad. Sci. U.S.A., 85:7079-7083 (1988), Zamecnik, et al., Proc. Natl. Acad. Sci. U.S.A., 83:4143-4146 (1986), and Goodchild, et al., Proc. Natl. Acad. Sci. U.S.A., 85:5507-5511 (1988); herpes simplex virus, Smith, et al., Proc. Natl. Acad. Sci. U.S.A., 83:2787-2791 (1986); and influenza virus, Zerial, et al., Nuc. Acids. Res., 15:9909-9919 (1987).
The concept underlying this work is that an oligonucleotide complementary to a unique segment of a viral genome, or an RNA derived from it, may selectively disrupt processes dependent on that segment by hybridization. This rationale is supported by a variety of experiments with cell free systems or with cells to which "antisense" polynucleotides have been inserted by microinjection or transfection, C. A. & Cohen, J. S., Cancer Res., 48:2659-2668 (1988). However, the actual mechanisms by which oligonucleotides and their analogs function as inhibitors in cell cultures are still far from clear. In particular, little is known about the interaction of the oligomers with cell membranes or the locus of their reactions within cells. It appears that non-ionic oligomers, such as the methyl phosphonate analogues diffuse passively through cell membranes.
S. E. Clare has synthesized oligonucleotides possessing one or more 2,2,2-trichloral-1,1-dimethylethyl (TDCME, lipophilic) group at the phosphorous atom in the chain and show that this group on one strand with proper stereochemistry can inhibit cleavage of the opposite strand by a restriction endonuclease and that the same group on a template will inhibit synthesis of the complementary strand by the Klenow enzyme. S. E. Clare also demonstrated a single modification 5' to dGNAd(CG) octamer by TDCME group prevents the B to Z conformational transition. S. E. Clare, Ph.D. Dissertation, Northwestern University, Evanston, Ill. (1987).
The present invention is related to a family of oligonucleotides modified at the backbone so that the oligonucleotide may anchor at the cell membrane to provide antiviral effects. The present invention describes a family of oligonucleotides with a modification designed to anchor the oligomer, at least transiently at the cell membrane, to inhibit HIV-1 in cell culture. Fatty substances have been selected as an anchor for the oligonucleotide, and without being limitative, cholesteryl has been selected as the preferred anchor since it is highly hydrophobic and cell membranes have an abundance of this steroid.
The cholesteryl is a large lipophilic group, much larger than the TDCME group. In principle, such pendent groups, when linked covalently to the internucleotide phosphorous atoms, have potential as lipophilic centers to enhance the interaction with membranes, to alter partitioning of oligonucleotides within cells, to inhibit certain enzymatic reactions and to influence the stability of hybrids joined with natural polynucleotides. Cholesteryl, is a component of many biological membranes and interacts with other lipids. The AIDS virus, HIV, is distinguished by an unusually high cholesteryl content in the lipid membrane. Early model studies by Finean, Experientia, 9:17-19 (1985), suggested that the cholesteryl molecule is capable of formation of a stabilizing complex with the phospholipid molecule. The hydrocarbon chain of the cholesteryl is bound to the parallel portion of the phospholipid chain by Van DeWall forces. Recent studies employing a variety of techniques indicated that the major forces may involve the hydrophobic portion of the lipid molecules. Therefore, cholesteryl is a preferred modifying group for oligonucleotide interaction with cells.